Comparison between bone–implant interfaces of microtopographically modified zirconia and titanium implants

The aim of this study was to investigate the surface characteristics and evaluate the bone–implant interfaces of injection molded zirconia implants with or without surface treatment and compare them with those of conventional titanium implants. Four different zirconia and titanium implant groups (n = 14 for each group) were prepared: injection-molded zirconia implants without surface treatment (IM ZrO2); injection-molded zirconia implants with surface treatment via sandblasting (IM ZrO2-S); turned titanium implants (Ti-turned); and titanium implants with surface treatments via sandblasting with large-grit particles and acid-etching (Ti-SLA). Scanning electron microscopy, confocal laser scanning microscopy, and energy dispersive spectroscopy were used to assess the surface characteristics of the implant specimens. Eight rabbits were used, and four implants from each group were placed into the tibiae of each rabbit. Bone-to-implant contact (BIC) and bone area (BA) were measured to evaluate the bone response after 10-day and 28-day healing periods. One-way analysis of variance with Tukey’s pairwise comparison was used to find any significant differences. The significance level was set at α = 0.05. Surface physical analysis showed that Ti-SLA had the highest surface roughness, followed by IM ZrO2-S, IM ZrO2, and Ti-turned. There were no statistically significant differences (p > 0.05) in BIC and BA among the different groups according to the histomorphometric analysis. This study suggests that injection-molded zirconia implants are reliable and predictable alternatives to titanium implants for future clinical applications.


Methods
Preparation of implant samples. A total of 56 screw-shaped dental implant samples (28 ZrO 2 implants (one-piece) and 28 Ti implants) of the same macroscopic shape and dimensions (a diameter of 3.4 mm and a length of 8 mm) were used in this study. Also, ten ZrO 2 discs, which were 15 mm in diameter and 1 mm in thickness, were prepared to find some phase transition of ZrO 2 after surface modification. The ZrO 2 implants were manufactured using an injection molding technique (Vatech Acucera, Seoul, Korea). This process involves mixing zirconia powders with modifiers and shaping a uniform and homogeneous mixture into a mold. The Ti implants were manufactured by a computer numerical controlled (CNC) milling technique (Deep Implant System, Inc., Seongnam, Korea). Disc-shaped green compacts were prepared by cold isostatic press of powder mixtures and then sintered. Surface modification with sandblasting was performed on half of the ZrO 2 implants and the ZrO 2 discs, while half of the Ti implant samples were surface-modified with SLA treatment, i.e., sandblasted with large-grit alumina (Al 2 O 3 ) particles and etched with hydrochloric acid (SLA surface; Deep Implant System, Inc., Seongnam, Korea). After sandblasting, the ZrO 2 implants (14 implants) and discs (5 discs) were treated by hot isostatic pressing (HIP) at 1380 °C and 138 MPa. Then, the samples were divided into four experimental groups: Scientific Reports | (2023) 13:11142 | https://doi.org/10.1038/s41598-023-38432-y www.nature.com/scientificreports/ (1) Group 1 = IM ZrO 2 (injection-molded ZrO 2 implants or ZrO 2 discs without sandblasting modification) (2) Group 2 = IM ZrO 2 -S (injection-molded ZrO 2 implants or ZrO 2 discs with sandblasting modification) (3) Group 3 = Ti-turned (Ti implants without SLA modification as a negative control) (4) Group 4 = Ti-SLA (Ti implants with SLA modification as a positive control) Assessment of surface characteristics. The implant sample surfaces were photographed by field emission-scanning electron microscopy (FE-SEM; S-4700, Hitachi, Tokyo, Japan). Surface parameters for the sample topography were measured by confocal laser scanning microscopy (CLSM; LSM 800, Carl Zeiss AG, Oberkochen, Germany). The acquired images were analyzed using ConfoMap software, and specific areas of interest were selected. Subsequently, the surface topography was quantified in terms of Sa (arithmetical mean height of a surface), the absolute value of the difference in height of each point compared to the arithmetical mean of the surface, and Sdr (developed interfacial area ratio), the proportion of the additional surface area contributed by the texture within the defined planar area. Each sample was analyzed at 3 selected sites (the upper, middle and lower flank), the values of which were averaged, and the average value was assigned as the representative value for the sample 22,33 . The topography was measured in terms of Sa values (arithmetical mean height) and Sdr values (developed interfacial area ratio). In addition, the chemical composition of each sample was analyzed with an energy-dispersive spectroscopy (EDS) device (EMAX, Horiba, High Wycombe, United Kingdom). For evaluating the phase transition of ZrO 2 , the ZrO 2 disc surfaces were analyzed using a high resolution X-ray diffractometer (SmartLab, Rigaku, Tokyo, Japan) with Cu Kα radiation (wavelength = 1.54 Å) and 45 kV/200 mA. To quantify the molar fraction of the content of monoclinic ZrO 2 ( X m ), the following equations were employed: where I t and I m represent the intensity of the tetragonal (101) and monoclinic (111) and 111 peaks 34,35 . The peak intensity was obtained using MDI Jade 6 software (Materials Data Inc., Livermore, CA, USA).
In vivo surgery. Eight male New Zealand white rabbits (age: 3-4 months old; weight: 2.5-3.0 kg) were used in this in vivo study, which was approved by the Institutional Animal Research Ethics Committee of Cronex (CRONEX-IACUC: 202108011, Hwaseong, Korea) and conducted according to the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines 36 . All methods were performed in accordance with the relevant guidelines and regulations. The experimental animals acclimatized in separate cages for two weeks before surgery. Each rabbit received four implant samples; two implants were placed in each tibia bone of the hind legs. The ZrO 2 and Ti implants were inserted based on the split-plot design (Fig. 1). For anesthesia, a combination of 15 mg/kg tiletamine hydrochloride and zolazepam hydrochloride (Zoletil 50; Virbac Korea Co., Ltd., Seoul, Korea) and 5 mg/kg xylazine (Rompun; Bayer Korea, Ltd., Seoul, Korea) was administered intramuscularly. Then, the hind legs were shaved and disinfected with an antiseptic surgical scrub of 7.5% povidone-iodine (Betadine; Korea Pharma, Seoul, Korea). After site preparation, local infiltration anesthesia of 2% lidocaine hydrochloride with 1:100,000 epinephrine (Yuhan Company, Seoul, Korea) was administered at the surgical sites. The tibial bones were exposed by full-thickness incisions from the skin to the periosteum. The surgical sites on the tibiae bones were prepared with rotating implant drills and engines under copious irrigation with sterile saline solution. The final drill size was 3.0 mm, and 32 sample implants from experimental groups 1, 2, 3 and 4 were placed with primary stability (≥ 20 Ncm) using a torque wrench, according to the manufacturer's instructions. After the implant placement surgeries, the muscle and fascia were sutured with resorbable 4-0 Vicryl sutures (Coated Vicryl; Ethicon, Raritan, NJ, United States), and the outer dermis was closed with nylon (Blue nylon; Ailee, Busan, Korea). All rabbit specimens were housed in individual cages and administered the postoperative antibiotic prophylaxis of enrofloxacin (Biotril, Komipharm International, Siheung, Korea).

Assessment of histology and histomorphometry.
Four rabbits were sacrificed 10 days after the implant placement (Rabbit 1-4), and the remaining 4 rabbits were sacrificed 28 days after the implant placement (Rabbit 5-8) by an overdose of potassium chloride intravenously under anesthesia for histologic assessment. The bones and connective tissues that surrounded the implant samples were surgically harvested en bloc. These blocks were fixed in 10% neutral buffered formalin for two weeks and then dehydrated with ethanol, followed by embedding in light-curing resin (Technovit 7200 VLC, Kulzer, Wehrheim, Germany). A series of cutting and grinding devices (EXAKT system; EXAKT Apparatebau, Norderstedt, Germany) was used to cut and grind the embedded blocks into slides with a thickness of less than approximately 50 μm 22,37 . The slides were stained with modified Goldner's Masson trichrome staining solution for examination under a light microscope. This staining technique allows for easy discrimination between newly formed bone (stained red) and exiting mature bone (stained blue) 38 . The bone-to-implant interfaces were measured by the degree of BIC and bone area (BA) at the best three consecutive threads 22,39 . The histologic evaluation was performed using a light microscope (DM2700M, Leica Microsystems CMS GmbH, Wetzlar, Germany) and an attached digital camera (DMC5400, Leica Microsystems CMS GmbH, Wetzlar, Germany). An image analysis system (ImageJ 1.60, NIH, Bethesda, MD, United States) was used to analyze the acquired images.
Statistical analysis. Most of the outcome variables for data normalization were accepted when the Shapiro-Wilk test was used (p > 0.05). Descriptive statistics are shown as the means and standard deviations (SDs).
One-way analysis of variance (ANOVA) was used to analyze differences in the mean values of the surface param- Consent for publication. All authors are aware of the publication of this work.

Results
Surface characteristics. The FE-SEM images of the implant surfaces of the 4 different experimental groups are shown in Fig. 2A. The IM ZrO 2 (Group 1) implants showed microcracks, porosities, and grain structures that are typically observed for sintered ZrO 2 . In contrast, the IM ZrO 2 -S (Group 2) implants showed very rough surfaces with slate-like profiles and lower porosities. The Ti-turned (Group 3: negative control) implants showed smooth and flat surfaces with continuous straight lines that ran in one direction. However, the Ti-SLA (Group 4: positive control) implants exhibited a rough, irregular, porous, and honeycomb-like appearance.
The surface roughness parameters of the experimental groups were measured in terms of Sa and Sdr values (Fig. 2B,C). Based on the mean Sa values and SDs, the Ti-SLA implants had the highest Sa value (1.68 μm ± 0.07 μm), followed by the IM ZrO 2 -S implants (1.10 μm ± 0.13 μm), IM ZrO 2 implants (0.64 μm ± 0.12 μm), and Ti-turned implants (0.52 μm ± 0.21 μm). There were statistically significant differences among the Sa values of all groups, except between the IM ZrO 2 and Ti-turned groups. The mean Sdr values of the Ti-SLA implants (238.78% ± 3.03%) were significantly higher than those of the other three groups. However, there were no significant differences among the Sdr values of these three groups: IM ZrO 2 (49.04% ± 31.68%), IM ZrO 2 -S (78.77% ± 50.02%), and Ti-turned (123.66% ± 37.54%).
The EDS findings are shown in Table 1. Titanium (Ti), carbon (C), and oxygen (O) were detected in the Ti implants, while zirconium (Zr), carbon (C), and oxygen (O) were detected in the ZrO 2 implants. The X-ray diffraction patterns of the ZrO 2 discs are shown in Fig. 3. ZrO 2 discs without sandblasting modification only presented the tetragonal ZrO 2 peak. ZrO 2 discs with sandblasting modification presented the (111) and 111 peaks for the monoclinic phase (m-ZrO 2 ) and the (101) peak for the tetragonal phase (t-ZrO 2 ). However, it was www.nature.com/scientificreports/ clearly seen that while the ZrO 2 discs with sandblasting modification presented both the peaks corresponding to the tetragonal and monoclinic phases, only the peak of tetragonal phase was detected after HIP. As seen in Table 2, the rate of the tetragonal-to-monoclinic phase transformation increased with sandblasting modification. It was found that the amount of m-ZrO 2 was 40.32% of the total ZrO 2 after sandblasting modification. After HIP, this amount of monoclinic phase was totally transformed into t-ZrO 2 .
Analysis of histology and histomorphometry. All the implant samples successfully osseointegrated after 10 days of healing and 28 days of healing ( Supplementary Fig. S1). In the histological analysis of the ZrO 2 and Ti implants stained with modified Goldner's Masson trichrome staining solution, new bone formation was found along the bone-implant interfaces. In the cortical bone area of the tibiae, the exiting mature bones were stained blue, while newly formed immature bones were stained red and detected at the implant threads and around the mature bone (Fig. 4). The mean BIC and BA values and SDs of the ZrO 2 and Ti implants at the 10-day and 28-day marks are shown in Fig. 5 (Supplementary Tables S1, S2). Although the BICs (%) of the IM ZrO 2 -S and Ti-SLA implants were higher than those of the Ti-turned and IM ZrO 2 implants at the 10-day mark, the differences were not statistically significant. At the 28-day mark, the BIC (%) of the Ti-SLA implants was significantly higher than that of the Ti-turned implants (p = 0.04). However, the other groups showed no significant differences. When the mean values and SDs of BA (%) were calculated, no statistically significant differences were found among the ZrO 2 and Ti implants for both healing periods (p > 0.05) (Fig. 5).

Discussion
The roughness parameters (Sa and Sdr) of the surface-treated implants (Groups 2 and 4) were significantly higher than those of the ZrO 2 and Ti implant groups without surface treatment (Groups 1 and 3). The authors also found that the BIC% and BA% of the IM ZrO 2 implants were not significantly different from those of the Ti implants (both Ti-turned and Ti-SLA) after 10-day and 28-day healing periods. These results are in agreement with the findings of previous studies that compared SLA-treated IM ZrO 2 and Ti implants in mini pig maxillae models 40 , canine models 41 , and rabbit tibia models 42 . Another significant finding was that the surface-treated ZrO 2 implants showed enhanced bone integration at the implant surface compared to the untreated ZrO 2 implants. This finding is in line with the results of other studies by Mihatovic in 2017 and Schünemann in 2019 43,44 . In addition, the Ti-SLA implants demonstrated a significantly higher bone response than the Ti-turned implants. This finding is also comparable to findings in other studies 45,46 . It can be deduced that the rough surface microtopography of www.nature.com/scientificreports/ ZrO 2 implants generated by the proper surface modification can influence early bone response and long-term sustainability.
In this study, the authors used 10-day and 28-day healing periods for bone formation in a rabbit tibia model based on previous studies 47,48 . These healing periods of rabbit specimens are equivalent to 1-month and 3-month healing periods of humans, according to Roberts, who demonstrated that the bone healing of rabbits was approximately three times faster than that of humans 49 . Although rabbits are regarded as commonly used and well-established animal models for investigating the osseointegration process, some disadvantages include site limitations and mismatched microstructures when compared to those of human bone. In addition, histomorphometric analysis has some disadvantages, although BIC and BA have become the most popular parameters. For example, they are only measured in 2 dimensions without consideration of the entire implant, and they may be affected by the quality and quantity of the surrounding bones 50 . There were slight discrepancies in the geometry of the ZrO 2 and Ti implants that might have affected the tissue reaction at the bone-implant interface. The small sample size employed in this study also presents a limitation. The absence of significant differences observed in the in vivo experiments could be attributed to this small sample size. Further studies are needed to develop methods for the predictable early bone response and long-term osseointegration of IM ZrO 2 implants with a double-blinded study design and a larger sample size. Furthermore, it is needed to investigate the optimal surface roughness of ZrO 2 implants for osseointegration in subsequent studies.

Conclusion
Based on the findings of this study, it is evident that appropriate surface treatment of ZrO 2 implants is essential for promoting early peri-implant bone formation. Considering the recognized advantages of simplicity, mass production capability, and economic feasibility associated with the injection molding technique, utilizing injection molded zirconia dental implants, coupled with suitable surface modifications, could present a promising alternative to conventionally manufactured titanium dental implants for future clinical applications.

Data availability
All data generated or analyzed during this study are included in this published article.